Method of separating protective components of bordetella pertussis

ABSTRACT

On the basis of differences in adsorbability to calcium phosphate gel formed by adding calcium ions to a  Bordetella pertussis  culture in the presence of excess phosphate ions, protective components of  Bordetella pertussis  are separated from the  Bordetella pertussis  culture.  
     Traditionally, protective components of  Bordetella pertussis  have been separated using different purification methods for the respective components. According to the present invention, the use of the same means of purification for all subject components makes it possible to purify each component with high efficiency and high recovery rate, an aspect very advantageous for industrial production. It is also possible to efficiently produce an improved purified pertussis component vaccine comprising an effective combination of pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT).

TECHNICAL FIELD

[0001] The present invention relates to a method of separatingprotective components of Bordetella pertussis. The pertussis componentvaccine can be produced by suitably mixing the protective componentsseparated by the method of the present invention.

BACKGROUND ART

[0002] Vaccines are widely used to prevent communicable diseases.Pertussis, a communicable respiratory disease caused by infection withBordetella pertussis, is likely to severely affect patients, especiallyinfants, due to apneic cough with occasional spasm. To cope with thisdisease, it has been common practice to use whole cultured cells ofBordetella pertussis after inactivation (inactivated vaccine). However,localized reactions at the site of vaccination and side reactions, suchas fever, have been reported, creating a social urge to solve thisproblem. To solve this problem, there have been a large number ofattempts of using protective components separated from Bordetellapertussis as vaccine. For example, acellular pertussis vaccine (ACPvaccine), prepared by extracting protective proteins, such as pertussistoxin (PT), pertussis filamentous hemagglutinin (FHA), pertactin (PRN,69K-OMP) and pertussis fimbriae (FIM), from Bordetella pertussis cells,and removing endotoxin (ET), is being into practical application, but isnot fully satisfactory, due to the drawbacks described below.

[0003] Pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA),pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM), all protectivecomponents of Bordetella pertussis already in practical application withvalidated efficacy, are separated by respective methods.

[0004] Pertussis toxin (PT) can be separated by affinity chromatographyusing human haptoglobin as a ligand [Biochimica et: Biophysica Acta,Vol. 580, p. 175 (1979)]. However, human haptoglobin can be contaminatedwith hepatitis virus, because it is collected from human blood; the sameapplies when animal sera are used. Another available method is affinitychromatography using denatured ceruloplasmin as a ligand (JapanesePatent Unexamined Publication No. 62135/1987). Although this method isfree of the problem of viral contamination, some problems arise,including vaccine contamination with ceruloplasmin and the high toxicityand potential body retention of sodium thiocyanate and other eluentshaving protein-denaturing effect.

[0005] As for pertussis filamentous hemagglutinin (FHA), a purificationmethod using hydroxyapatite gel is available [Infection and Immunity,Vol. 41, p. 313 (1983) and EP-A-231083, EP-A-427462, EP-A-462534;Japanese Patent Unexamined Publication Nos. 234031/1987, 169893/1992,368337/1993). However, it takes long time for column operation, and isuneconomic due to the high cost of hydroxyapatite.

[0006] As for pertactin (PRN, 69K-OMP), affinity chromatography using amouse serum as a ligand is available [Infection and Immunity, Vol. 56,p. 3189 (1988)], but has the same drawbacks as above.

[0007] As for pertussis fimbriae (FIM), Bordetella pertussis cellextract is purified by salting-out with ammonium sulfate and magnesiumchloride [Infection and Immunity, Vol. 48, p. 442 (1985)], but thismethod is poor in vaccine production efficiency due to low yield.

[0008] There is a method of preparing Gram-negative bacterial vaccine byadosorbing with the aluminum hydroxide gel (WO 93/10216). This methodneeds the large amount of the aluminum hydroxide gel, which adsorbs boththe protective components and the endotoxin originated in Gram-negativebacteria. The vaccine obtained by the method of WO93/10216 has a dangerof side effects, such as fever and endotoxin-shock, by the endotoxinreleased into body because of the diluted vaccines.

[0009] As for the pertussis vaccine production included as thecomponents mixture without separating each protective componentoriginated in Bordetella pertussis, a method of using calcium phosphategel is available (EP-A-291968, Japanese Patent Unexamined PublicationNo. 52726/1989). However, this method formed the calcium phosphate inthe presence of a 1M sodium chloride does not absorb the protectivecomponents.

[0010] As stated above, totally different purification methods must beused to separate the respective protective components of Bordetellapertussis. This approach is unsuitable to large-scale vaccine productiondue to painstaking operation, and difficult to apply practically.Moreover, the customary methods of separating protective componentsdisclosed in prior art have some problems that materials or reagentshave pathogenicity or toxity.

DISCLOSURE OF INVENTION

[0011] Against the background described above, the present inventorsinvestigated methods of efficiently separating protective components ofBordetella pertussis, and found that protective components of Bordetellapertussis can be efficiently separated from Bordetella pertussis cultureon the basis of differences in adsorbability to calcium phosphate gelformed by adding calcium ions to the Bordetella pertussis culture in thepresence of excess phosphate ions. The inventors made furtherinvestigation based on this finding, and the efficient and safty methodof separating the protective components combined with the calciumphosphate gel treatment and elution by salt and heating was developedthe present invention. Accordingly, the present invention relates to:

[0012] (1) A method of separating at least one member of the groupconsisting of pertussis filamentous hemagglutinin (FHA), pertactin (PRN,69K-OMP), pertussis fimbriae (FIM), and pertussis toxin (PT) by bringinga Bordetella pertussis culture into contact with calcium phosphate gelwhich is formed by adding calcium ions to the culture in the presence ofphosphate ions.

[0013] (2) A method of separating at least one member of the groupconsisting of pertussis filamentous hemagglutinin (FHA), pertactin (PRN,69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT) byseparating a Bordetella pertussis culture into cells and culture liquid,and carrying out at least one of processes (A), (B), (C) and (D):

[0014] (A) a process in which the separated cells are eluted with a saltsolution, and pertussis filamentous hemagglutinin (FHA) is separated bybringing the eluted solution into contact with calcium phosphate gel ofthe above item (1),

[0015] (B) a process in which the cell residue resulting from theelution treatment of the above process (A) is heated in the presence ofa salt solution and brought into contact with calcium phosphate gel, andpertactin (PRN, 69K-OMP) is separated by bringing the eluted solutioninto contact with calcium phosphate gel of the above item (1),

[0016] (C) a process in which the cell residue resulting from theelution treatment of the above process (A) is heated in the presence ofa salt solution, the supernatant is brought into contact with calciumphosphate gel and eluted with a salt solution, and pertussis fimbriae(FIM) is separated by bringing the eluted solution into contact withcalcium phosphate gel of the above item (1),

[0017] (D) a process in which the culture or the separated cultureliquid is brought into contact with calcium phosphate gel of the aboveitem (1), and pertussis toxin (PT) is separated from the supernatant.

[0018] (3) The separation method of the above item (2), wherein thesupernatant is brought into contact with calcium phosphate gel andeluted with a salt solution to separate pertussis filamentoushemagglutinin (FHA) in process (A).

[0019] (4) The separation method of the above item (2), wherein thesupernatant after being brought into contact with calcium phosphate gelis brought into contact with ion exchange gel to separate pertactin(PRN, 69K-OMP) in process (B).

[0020] (5) The separation method of the above item (2), wherein thesupernatant is brought into contact with calcium phosphate gel andremoved, and the resulting residue is eluted with a salt solution toseparate pertussis fimbriae (FIM) in process (C).

[0021] (6) The separation method of the above item (2), wherein thesupernatant is brought into contact with ion exchange gel to separatepertussis toxin (PT) in process

[0022] (7) The separation method of the above item (2), wherein the saltsolution used in processes (A) and (C) is a buffer containing an alkalimetal salt.

[0023] (8) The separation method of the above item (7), wherein the saltsolution is a buffer containing 0.01-1.0 M sodium chloride.

[0024] (9) The separation method of the above item (1) or (2), whereinthe calcium phosphate gel is formed by adding calcium ions to theculture or the supernatant of pH 7-9 in the presence of phosphate ions.

[0025] (10) The separation method of the above item (9), wherein theequivalent ratio of phosphate ions and calcium ions is 1.25-30equivalents of phosphate ions per equivalent of calcium ions.

[0026] (11) The separation method of the above item (9), wherein thecalcium phosphate gel is formed by adding calcium acetate, as a calciumion source, at 0.1-2 w/v% in the presence of a 0.05-0.1 M phosphatebuffer.

[0027] (12) The separation method of the above item (1) or (2), whereinat least one member of the group consisting of pertussis toxin (PT),pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) andpertussis fimbriae (FIM) is separated, after which endotoxin is removedby adsorption to aluminum hydroxide gel in the presence of ammoniumsulfate.

[0028] (13) The separation method of the above item (1) or (2), whereinat least one member of the group consisting of pertussis toxin (PT),pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) andpertussis fimbriae (FIM) is separated, after which endotoxin is removedby zonal centrifugation.

[0029] (14) A pertussis vaccine wherein the components PT:FHA:FIM areadmixed in a ratio of 4-6:8-10:1.

[0030] (15) A pertussis vaccine wherein the components PT:FHA:PRN:FIMare admixed in a ratio of 2-6:4-10:1-2:1.

BEST MODE FOR CARRYING OUT THE INVENTION

[0031] The Bordetella pertussis strain used for the present invention isnot subject to limitation, as long as it is capable of producing one ormore than one member of the group consisting of pertussis filamentoushemagglutinin (FHA), pertactin (PRN, 69K-OMP), pertussis fimbriae (FIM)and pertussis toxin (PT), all protective components of Bordetellapertussis. Useful strains include known strains, such as Bordetellapertussis Tohama phase I strain (Infection and Immunity, Vol. 6, p. 89,(1972)] (maintained at the National Institute of Health, Ministry ofSocial Welfare, Tokyo, Japan (NIHJ 1052), deposited under accessionnumber IFO 14073 at the Institute for Fermentation, Osaka since Aug. 13,1980), Bordetella pertussis Yamaguchi phase I strain, Bordetellapertussis phase I strain 18-323 and Bordetella pertussis phase I strain165, with preference given to Bordetella pertussis Tohama phase I strain(IFO 14073) from the viewpoint of productivity. Bordetella pertussis canbe cultured by known methods. Useful media include known basal media,such as Cohen-Wheeler medium, Stainer-Scholte medium and other liquidmedia, with preference given to Stainer-Scholte medium. The solutioncontaining protective components and endotoxin (ET) may be a cultureobtained by stationary culture or tank culture. In the presentinvention, the culture means cultured cells or culture liquid resultingfrom incubating said Bordetella pertussis. And the present invention,the supernatant means the culture liquid or the supernatant resultingfrom heating the cells in the presence of a salt solution or elutingwith a salt solution from the calcium phosphate gel adsorbed theprotective components as described below. The cells include the culturecells and the cell residue. In the present invention, the method used toseparate a Bordetella pertussis culture into cells and culturesupernatant may be a known method, such as centrifugation or filtration.

[0032] The calcium phosphate gel used for the present invention is not aready-made gel, but preferably calcium phosphate gel formed in a cultureor a supernatant to be treated by adding calcium ions to them in thepresence of excess phosphate ions (may referred to as the in-side gelforming method). Although prepared calcium phosphate gel (e.g.,commercially available hydroxyapatite gel). In comparison with theformer method by using the ready-made hydroxyapatite gel mentioned above(may referred to as the out-side gel forming method), the present methodby using calcium phosphate gel is higher in both adsorption efficiencyfor pertussis filamentous hemagglutinin (FHA) and pertussis fimbriae(FIM) and recovery efficiency of them, as shown hereafter. Moreover, thecalcium phosphate gel used in the present invention is better inoperational efficiency because of the absence of gel pretreatment andregeneration process, and more advantageous in cost. Furthermore, eachof protective components of Bordetella pertussis can be selectivelyabsorbed to the calcium phosphate gel by properly selecting the ratio ofphosphate ions to calcium ions. If the the culture or the supernatant tobe treated with calcium phosphate gel, does not contain a sufficientamount of phosphate ions, a phosphate buffer of appropriateconcentration is added to provide phosphate ions before addition ofcalcium ions. For example, by adding 1 M phosphate buffer, the finalphosphate ion, concentration is adjusted to 0.02-0.2 M, preferably0.05-0.1 M.

[0033] The calcium ion source added is exemplified by soluble calciumsalts,, such as calcium acetate, calcium chloride and calcium nitrate,with preference given to calcium ions derived from calcium acetate.Concerning the ratio of phosphate ions and calcium ions, it ispreferable that phosphate ions be in excess, in comparison with calciumions. The ratio can be properly selected in each case of the protectivecomponents of Bordetella pertussis, as mentioned hereafter.

[0034] In process (A) above, pertussis filamentous hemagglutinin (FHA)is separated as follows: After the culture liquid, i.e. the culturesupernatant, is removed from a Bordetella pertussis culture by a knownmethod, such as centrifugation or filtration, a one-tenth toone-twentieth volume (relative to the amount of culture broth)(corresponding to a final cell concentration of 50-100 billion cells/ml)of a salt solution is added to the cells to elute the hemagglutinin. Inthis case, the salt solution used is preferably a buffer supplementedwith an alkali metal salt or an alkaline earth metal salt, specificallya 0.04-0.08 M phosphate buffer supplemented with a 0.25-1.0 M alkalimetal salt or alkaline earth metal salt, with greater preference givento a 0.05 M phosphate buffer supplemented with a 0.5-1.0 M alkali metalsalt. The alkali metal salt or alkaline earth metal salt added to thebuffer is exemplified by sodium chloride, potassium chloride andmagnesium chloride. For example, it is preferable to elute thehemagglutinin by adding a one-tenth to one-twentieth volume (relative tothe amount of culture broth) of a 0.04-0.08 M phosphate buffer (pH 7-9)supplemented with 0.5-1.0 M sodium chloride, more preferably a 0.05 Mphosphate buffer (pH 8) supplemented with 1 M sodium chloride, to thecells collected, followed by gentle stirring at 4° C. to roomtemperature, preferably 8-15° C., for 1-60 minutes, preferably 1-30minutes, and standing for 1-2 days. The solution containing the elutedpertussis filamentous hemagglutinin (FHA) is then subjected to a knownmethod, such as centrifugation or filtration, to recover the supernatant(the cell residue obtained at the same time by this treatment is used toisolate pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM)). Thethus-obtained supernatant is then brought into contact with calciumphosphate gel.

[0035] Concerning the ratio of phosphate ions and calcium ions, it ispreferable that phosphate ions be in excess, in comparison with calciumions. For example, the equivalent ratio of these ions is preferably1.25-30 equivalents, more preferably 1.5-7.5 equivalents of phosphateions per equivalent of calcium ions. This quantitative ratio can beexpressed in molar ratio as 0.8-20 M of phosphate ions to 1 M of calciumions, more preferably 1-5 M of phosphate ions to 1 M of calcium ions.For example, to a solution (pH 7-9) containing phosphate ions at aconcentration within the above-described concentration range (0.02-0.2M, preferably 0.05-0.1 M), a calcium salt is added to a finalconcentration of 4-70 mM, preferably 8-50 mM (e.g., calcium acetateadded to a final concentration of 0.1-0.8 w/v%, preferably 0.2-0.6w/v%), followed by gentle reaction at 4° C. to room temperature,preferably 8-15° C., for 1 to 4 hours, preferably 1 to 2 hours, to formcalcium phosphate gel.

[0036] Although pertussis filamentous hemagglutinin (FHA) is adsorbed tothe calcium acetate gel added, provided that the amount of calciumphosphate gel added to a final concentration exceeding 0.8 w/v%, it ispreferable to add the calcium acetate gel in an amount such that thefinal concentration falls within the above concentration range, forselectively adsorbing pertussis filamentous hemagglutinin (FHA) only.After completion of the reaction, the supernatant is removed by a knownmethod, such as centrifugation or filtration; the resulting gelprecipitate is collected. To this precipitate, a one-tenth toone-twentieth volume (relative to the amount of culture broth) of a saltsolution is added, to elute the pertussis filamentous hemagglutinin(FHA). In this case, the salt solution used may be the same saltsolution as used to elute pertussis filamentous hemagglutinin (FHA) fromthe above-described cells. It is preferable to add a one-tenth toone-twentieth volume of 0.05-0.1 M phosphate buffer (pH 7-9)supplemented with 1-2 M sodium chloride, more preferably 0.1 M phosphatebuffer (pH 8) supplemented with 1-1.5 M sodium chloride, to theabove-described gel precipitate, followed by gentle stirring at 4° C. toroom temperature for 1 to 2 hours, to elute the hemagglutinin. Aftercompletion of the stirring, the precipitate is removed by a knownmethod, such as centrifugation or filtration, to recover pertussisfilamentous hemagglutinin (FHA) in the supernatant. The supernatant, ifnecessary, can be concentrated and desalinized, by ammonium sulfatesalting-out or using an ultrafiltration membrane. By subjecting thesupernatant obtained by the above-described treatment to the aluminumhydroxide gel treatment or zonal centrifugation described below,pertussis filamentous hemagglutinin (FHA) having endotoxin selectivelyremoved can be separated with substantially no loss.

[0037] In process (B) or (C) above, pertactin (PRN, 69K-OMP) andpertussis fimbriae (FIM) are separated as follows: The cell residueresulting from elution of the solution containing pertussis filamentoushemagglutinin (FHA) is heated in the presence of a one-tenth toone-twentieth volume (relative to the amount of culture broth)(corresponding to a final cell concentration of 500-100 billioncells/ml) of a salt solution to extract the pertactin (PRN, 69K-OMP) andpertussis fimbriae (FIM). In this case, the salt solution used may bethe same as used in process (A) above. However, it is preferable to usea one-tenth to one-twentieth volume (relative to the amount of culturebroth) of 0.01-0.05 M phosphate buffer (pH 7-9) supplemented with0.15-0.25 M sodium chloride, with greater preference given to 0.01 Mphosphate buffer (pH 7) supplemented with 0.15-0.25 M sodium chloride.It is preferable that heating be achieved in warm water at 40-80° C.,preferably 50-60° C., for 60 to 120 minutes, preferably 80 to 90minutes. The heated extracted pertactin (PRN, 69K-OMP) and pertussisfimbriae (FIM) are recovered in the supernatant by a known method, suchas centrifugation or filtration. The thus-obtained supernatant is thenbrought into contact with calcium phosphate gel. In this case, calciumphosphate gel treatment can be performed in accordance with process (A)above; however, it is preferable to perform it within the followingconcentration range. For example, to a solution (pH 7-9) containingphosphate ions, adjusted as necessary to a final phosphate ionconcentration of 0.05-0.1 M, preferably 0.1 M, by adding a 1 M phosphatebuffer, or the like, a calcium salt is added to a final concentration of40-180 mM, preferably 55-150 mM (e.g., calcium acetate added to a finalconcentration of 1-2 w/v%, preferably 1.3-1.7 w/v%), followed by gentlereaction at 4° C. to room temperature, preferably 8-L5° C., for 1 to 4hours, preferably 1 to 2 hours, to form calcium phosphate gel. Aftercompletion of the reaction, the resulting precipitate and supernatantare separated from each other by a known separation method, such asfiltration or centrifugation, to recover pertactin (PRN, 69K-OMP) in thesupernatant and pertussis fimbriae (FIM) in the gel residue, withsubstantially no loss.

[0038] The crudely purified pertactin (PRN, 69K-OMP) obtained by theabove-described treatment can be further purified by a known method,preferably by ion exchange gel treatment; it is preferable that thecrudely purified pertactin (PRN, 69K-OMP) be previously concentrated anddesalinized by ammonium sulfate salting-out or using an ultrafiltrationmembrane. In the present invention, useful ion exchange gels includeanion exchange gel and cation exchange gel, with preference given tocation exchange gel. Contact with ion exchange gel may be achieved bythe column chromatography method or the batch method. By this treatment,impurities, i.e., substances other than pertactin (PRN, 69K-OMP) in thecrudely purified pertactin (PRN, 69K-OMP), are adsorbed; the effluent iscollected to yield a solution containing pertactin (PRN, 69K-OMP). Inthe column chromatography method, the column is packed with ion exchangegel, through which the starting material, i.e., crudely purifiedpertactin (PRN, 69K-OMP), is passed at a flow rate of 100-500 ml/cm²/hr.In the batch method, crudely purified pertactin (PRN, 69K-OMP) is placedin a container, to which ion exchange gel is added directly, followed bystirring for about 30 minutes to 3 hours, preferably about 1 hour, toadsorb impurities, i.e., substances other than pertactin (PRN, 69K-OMP).Such impurity adsorption is achieved using a buffer of a pH value of5.0-8.0 and an electroconductivity of 100-300 umho (0.1-0.3 mS), e.g., a0.01-0.02 M phosphate buffer (pH 5.5-6.0). By subjecting the supernatantobtained by the above-described treatment to the aluminum hydroxide geltreatment or zonal centrifugation treatment described below, pertactin(PRN, 69K-OMP) having endotoxin removed can be separated withsubstantially no loss.

[0039] To the gel residue containing crude pertussis fimbriae (FIM)obtained by the above-described treatment, a one-tenth to one-twentiethvolume (relative to the amount of culture broth) of a salt solution isadded, to elute the pertussis fimbriae (FIM). In this case as well, thesalt solution may be the same as used in process (A) above. For example,it is preferable to add a one-tenth to one-twentieth volume (relative tothe amount of culture broth) of a 0.05-0.1 M phosphate buffer (pH 7-9)supplemented with 1-2 M sodium chloride, preferably a 0.1 M phosphatebuffer (pH 8) supplemented with 1-1.5 M sodium chloride, followed bygentle stirring at 4° C. to room temperature for 1 to 2 hours, to elutethe pertussis fimbriae (FIM). After completion of the stirring, theprecipitate is removed by a known method, such as centrifugation orfiltration, to recover pertussis fimbriae (FIM) in the supernatant. Bysubjecting the supernatant obtained by the above-described treatment tothe above-described aluminum hydroxide gel treatment or zonalcentrifugation treatment, pertussis fimbriae (FIM) having endotoxinselectively removed can be separated with substantially no loss.

[0040] In process (D) above, pertussis toxin (PT) is separated asfollows: Although a Bordetella pertussis culture can be used withoutseparation into cultured cells and culture supernatant in this process,it is preferable in respect of efficiency to recover the supernatantfrom the Bordetella pertussis culture by a known method, such ascentrifugation or filtration, concentrate the supernatant about 10-20fold using an ultrafiltration membrane, or the like, and collect thesupernatant by centrifugation or another method before this process.This supernatant is then brought into contact with calcium phosphategel. In this case, calcium phosphate gel treatment can be carried out inthe same manner as in process (A) above, but it is preferable to carriedout this treatment within the following concentration range. Forexample, to a solution (pH 7-9) containing phosphate ions, adjusted asnecessary to a final phosphate ion concentration of 0.05-0.1 M,preferably 0.1 M, by adding a 1 M phosphate buffer, or the like, acalcium salt is added to a final concentration of 40-180 mM, preferably55-150 mM (e.g., calcium acetate added to a final concentration of 1-2w/v%, preferably 1.3-1.7 w/v%), followed by gentle reaction at 4° C. toroom temperature, preferably 8-15° C., for 1 to 4 hours, preferably 1 to2 hours, to form calcium phosphate gel. After completion of thereaction, the resulting precipitate and supernatant are separated fromeach other by a known method, such as centrifugation or filtration, torecover pertussis toxin (PT) in the supernatant with substantially noloss. The crudely purified pertussis toxin (PT) obtained by theabove-described treatment is further purified by ion exchange geltreatment; it is preferable that the crudely purified pertussis toxin(PT) be previously concentrated and desalinized by ammonium sulfatesalting-out or using an ultrafiltration membrane. The ion exchange gelused here is exemplified by anion exchange gel and cation exchange gel,with preference given to cation exchange gel. Contact with ion exchangegel may be achieved by the column chromatography method or the batchmethod. By this treatment, pertussis toxin (PT) in the crudely purifiedpertussis toxin (PT) is adsorbed to the gel, followed by washing with anappropriate buffer to elute and remove impurities, after which pertussistoxin (PT) is eluted and isolated with a buffer of appropriate pH andionic strength. In the column chromatography method, the column ispacked with ion exchange gel, through which the starting material, i.e.,crudely purified pertussis toxin (PT), is passed at a flow rate of100-500 ml/cm²/hr to cause toxin adsorption. In the batch method, thecrudely purified pertussis toxin (PT) is placed in a container, to whichion exchange gel is added directly, followed by stirring for about 30minutes to 3 hours, preferably about 1 hour, to cause toxin adsorption.Such adsorption of the crudely purified pertussis toxin (PT) is achievedusing a buffer of a pH level of 5.0-6.0 and an electroconductivity of100-300 umho (0.1-0.3 mS), e.g., a 0.01-0.02 M phosphate buffer (pH5.5-6.0). Elution from the ion exchange gel to which the pertussis toxin(PT) has been adsorbed can be achieved using a buffer of a pH level of7.0-7.5 and an electroconductivity of 1,000-2,000 umho (1-2 mS), e.g., a0.1-0.2 M phosphate buffer (pH 7.0-7.5). By subjecting the eluateobtained by the above-described treatment to the aluminum hydroxide geltreatment or zonal centrifugation treatment described below, pertussistoxin (PT) having endotoxin selectively removed can be separated withsubstantially no loss.

[0041] In the present invention, the aluminum hydroxide gel treatmentfor endotoxin removal is carried out to adsorb only the endotoxinselectively by bringing the subject into contact with previouslyprepared aluminum hydroxide gel in the presence of ammonium sulfate.But, the aluminum hydroxide gel, whose amount to be used is less thanone-tenth of that used in WO93/10216, hardly absorbs any amounts ofprotective components of Bordetella pertussis. It is normally preferablethat this treatment be carried out after concentration by a knownmethod, such as ammonium sulfate salting-out or an ultrafiltrationmembrane method. Aluminum ions useful for the previously preparedaluminum hydroxide gel include those of soluble aluminum compounds, suchas aluminum sulfate and aluminum chloride, with preference given to thealuminum ions of aluminum chloride. It is preferable that aluminumhydroxide gel be prepared by adding a 2 M sodium hydroxide solution to a25-190 mM aluminum salt solution (e.g., 0.9-4.5% aluminum chloridesolution) to a pH level of 7.0-7.5, followed by gentle reaction at 4° C.to room temperature for 1 to 3 hours, to form the desired aluminumhydroxide gel. The aluminum hydroxide gel obtained by theabove-described treatment is then treated to recover the resulting gelprecipitate by a known method, such as filtration or centrifugation, toremove free aluminum ions after completion of the reaction. Theprotective component of Bordetella pertussis concentrated by a knownmethod, such as ammonium sulfate salting-out, is recovered bycentrifugation; the precipitate is dissolved in a 0.25 M phosphatebuffer (pH 7.0-7.5) supplemented with 0.25 M sodium chloride. To thisprotective component of Bordetella pertussis, a saturated ammoniumsulfate solution is added to a final concentration of 2.0-8.0 v/v%,followed by addition of previously prepared, recovered aluminumhydroxide gel to a final concentration of 0.1-1.0 mg/ml, preferably0.2-0.5 mg/ml, and gentle reaction at 4° C. to room temperature for 30minutes to 1 hour. After completion of the reaction, the aluminumhydroxide gel is removed by a known method, such as filtration orcentrifugation, to separate the protective component of Bordetellapertussis having endotoxin removed, with substantially no loss.

[0042] In the present invention, zonal centrifugation treatment iscarried out to remove endotoxin, and is preferably carried out afterconcentration by a known method, such as ammonium sulfate salting-out.Zonal centrifugation methods include sucrose density gradientcentrifugation, cesium chloride density gradient centrifugation andpotassium tartrate density gradient centrifugation, with preferencegiven to sucrose density gradient centrifugation. For example, whensucrose density gradient centrifugation is carried out on a sucrosedensity gradient of 0-30 w/v% at an Rmax of 60,000 to 122,000 G forabout 10 to 24 hours, the protective component of Bordetella pertussishaving endotoxin removed can be separated.

[0043] PT is detoxified by using a conventional detoxification techniqueas described in British Journal of Experimental Pathslogy, vol. 44, p.177, (1963). FHA, PRN and FIM may be inactivated, for example, by themethod as described in Japanese Patent Unexamined Publication No.52726/1989. An improved purified pertussis component vaccine which issuperior to a known pertussis vaccine can be produced by blending in anydesireded ratio of protective components of Bordetella pertussisobtained by the method of present invention. Namely, it's not possibleto change the ratio of each component which is stable in whole cell orco-purified acellular vaccine without obtaining furified componentrespectively, while an antigen ratio can be chosen in the method ofpresent invention which gives the optimal which gives the optimalresponse in humans as a pertussis vaccine since each component isefficiently purified in the present invention. The purified pertussiscomponent vaccine is desirable to blend the protective components in aslittle amount of total protein as possible and in a way of giving moreeffective immunogenicity. The purified pertussis component vaccine ofthe present invention preferably includes all of three components, i.e.FHA, FIM and PT, and may also include other pharmaceutically acceptablecomponents such as PRN which does not give undesired side effects.

[0044] When blending these components to produce a purified pertussiscomponent vaccine of the present invention, the ratio of it may beexamplified in Examples metioned hereinafter. The component vaccine ofthe present invention has a PT:FHA:FIM ratio of approximate 4-6:8-10:1,preferably 5-6:8-10:1, and comprise, for example, 20-30 μg-protein/ml ofPT, 40-50 μg-protein/ml of FHA and 5-10 μg-protein/ml of FIM, preferably25-30 μg-protein/ml of PT, 40-50 μg-protein/ml of FHA and 5μg-protein/ml of FIM. The component vaccine mentioned above may furtherinclude 5-10 μg-protein/ml of PRN, and has a PT:FHA:PRN:PT ratio of2-6:4-10:1-2:1, preferably 5-6:8-10:2:1. Namely, it preferably comprise25-30 μg-protein/ml of PT, 40-50 μg-protein/ml of FHA, 10 μg-protein/mlof PRN and 5 μg-protein/ml of FIM.

[0045] The above-described effect of the present invention can besummarized as follows: The method of the present invention ischaracterized by the use of the same means of purification for allsubject protective components of Bordetella pertussis. This obviates thenecessity of different painstaking procedures for the respectivecomponents as in prior art methods, thus permitting componentpurification with high efficiency and high recovery rate, an aspect veryadvantageous for industrial production. In addition, the endotoxincontent, as determined by the Limulus test, is not more than 1 ng per100 μg total protein, for all protective components of Bordetellapertussis obtained by the present invention, providing very highpractical value. It is also possible to produce an improved purifiedpertussis component vaccine comprising an effective combination ofpertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP),pertussis fimbriae (FIM) and pertussis toxin (PT).

EXAMPLES

[0046] The present invention is hereinafter described in more detail bymeans of, but is not limited to, the following working examples andreference examples. In the following description, pertussis toxin (PT),pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP),pertussis fimbriae (FIM) and endotoxin are also referred to as PT, FHA69K-OMP, FIM and ET, respectively.

Example 1

[0047]Bordetella pertussis Tohama phase I strain was cultured to a finalconcentration of 2 billion cells/ml by Roux bottle stationary culture(450 ml, 35° C., 5 days) and tank agitating culture (40 l, 35° C., 2days) using Stainer-Scholte medium, to yield a Bordetella pertussisculture.

[0048] The cell culture was concentrated to a one-tenth volume using anultrafiltration membrane, after which it was centrifuged to separate thesupernatant and cells. To the supernatant, a 1 M phosphate buffer (pH8.0) was added to a final concentration of 0.1 M, followed by additionof an calcium acetate solution to a final concentration of 1.6 w/v% andstirring at room temperature for 1 hour. This calcium phosphate gelsolution was filtered. The resulting filtrate was concentrated anddesalinized to an electroconductivity of 200 umho using anultrafiltration membrane, passed through a sulfopropyl cation exchangechromatography column (produced by Tosoh Corporation), washed with a0.01 M phosphate buffer (pH 6.0), and eluted with a 0.1 M phosphatebuffer (pH 7.0), to yield pertussis toxin (PT). Next, cells weredispersed in a one-tenth volume (relative to the amount of culturebroth) of a 0.05 M phosphate buffer (pH 8.0) supplemented with 1 Msodium chloride, followed by centrifugation to yield the supernatant andcells. To the supernatant, a calcium acetate solution was added to afinal concentration of 0.5 w/v%, followed by stirring at roomtemperature for 1 hour. This calcium phosphate gel solution wasfiltered; the resulting gel layer was collected. The gel layer waseluted with a 0.1 M phosphate buffer (pH 8.0) supplemented with 1 Msodium chloride to yield a solution containing pertussis filamentoushemagglutinin (FHA). Separately, cells were dispersed in a one-tenthvolume (relative to the amount of culture broth) of a 0.01 M phosphatebuffer (pH 7.0) supplemented with 0.15 M sodium chloride, after which itwas heated in 60° C. warm water for 90 minutes, followed bycentrifugation to yield the supernatant. To the supernatant, a 1 Mphosphate buffer (pH 8.0) was added to a final concentration of 0.1 M,after which a calcium acetate solution was added to a finalconcentration of 1.6 w/v%, followed by stirring at room temperature for1 hour. This calcium phosphate gel solution was filtered; the filtrateand the gel layer were collected. The filtrate was concentrated anddesalinized to an electroconductivity of 200 umho using anultrafiltration membrane and passed through a sulfopropyl cationexchange chromatography column (produced by Tosoh Corporation); theeffluent was collected to yield a solution containing pertactin (PRN,69K-OMP). Separately, the gel layer was eluted with a 0.1 M phosphatebuffer (pH 8.0) supplemented with 1 M sodium chloride to yield asolution containing pertussis fimbriae (FIM).

[0049] Control sample was prepared as follows: Ammonium sulfate wasadded at 220 g per liter of culture broth, followed by sufficientstirring. After being kept standing at 4° C. for about 14 days, themixture was centrifuged; the supernatant was discarded, and theprecipitate was collected. To the precipitate thus obtained, a one-tenthvolume (relative to the amount of culture broth) of a 0.05 M phosphatebuffer (pH 8.0) supplemented with 1 M sodium chloride was added,followed by sufficient stirring. After being kept standing at 4° C. for4 days, the mixture was again centrifuged; the supernatant was collectedto yield a solution containing pertussis toxin (PT), pertussisfilamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) or pertussisfimbriae (FIM).

[0050] The pertussis toxin (PT), pertussis filamentous hemagglutinin(FHA), pertactin (PRN, 69K-OMP) or pertussis fimbriae (FIM) content ineach sample was determined by ELISA, with purified products of pertussistoxin (PT), pertussis filamentous hemagglutinin (FHA), pertactin (PRN,69K-OMP) and pertussis fimbriae (FIM) as references. Results areexpressed in μg protein/ml unit.

[0051] Protein content determination: Protein precipitated with heatedtrichloroacetic acid was quantitated by the Lowry method, with bovineserum albumin (Fraction V, produced by Wako Pure Chemical Industries) asa reference. Results are expressed in μg protein/ml unit.

[0052] The results for Roux bottle culture broth and those for tankculture broth are shown in Tables 1 and 2, respectively. TABLE 1 ActivePurity (%) Ingredient Total (active Protein Protein ingredient proteinContent (μg Recovery* Content content/total Sample protein/ml) (%) (μgprotein/ml) protein content) PT 2656.8 90.0 2662.1 99.8 FHA 9161.7 85.09339.1 98.1 FIM 474.7 244.6 495.5 95.8 69K-OMP 3683.8 244.6 3607.2 102.1

[0053] TABLE 2 Active Purity (%) Ingredient Total (active ProteinProtein ingredient protein Content (μg Recovery* Content content/totalSample protein/ml) (%) (μg protein/ml) protein content) PT 3242.2 78.93359.8 96.5 FHA 9527.0 98.0 9752.9 97.7 FIM 675.0 1184.0 714.7 95.069K-OMP 4333.5 2364.0 4505.1 96.3

[0054] It is evident from these figures that each protective componentwas efficiently isolated, and that in the case of tank culture broths,pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM), both produced atlow productivity in the case of Roux bottle culture broths, wererecovered in large amounts.

Reference Example 1

[0055] To a control solution prepared by the method described in Example1, calcium acetate was added to a final concentration of 0.5 w/v%,followed by stirring at room temperature for 1 hour. To the filtrateobtained by filtering this; calcium solution, a half amount of asaturated ammonium sulfate solution was added; the mixture was keptstanding at 4° C. for 7 days. This ammonium sulfate salting-out productwas centrifuged; the resulting precipitate was collected and resuspendedin a 0.025 M phosphate buffer (pH 7.0) supplemented with 0.25 M sodiumchloride to yield a starting material. To this starting material,aluminum hydroxide gel, previously prepared to a final concentration of0.4 mg/ml, was added; to the aluminum hydroxide gel recovered bycentrifugation, ammonium sulfate was added to a final concentration of0, 2, 4 or 8 w/v%, followed by gentle stirring at room temperature for30 minutes. After completion of the reaction, the aluminum hydroxide gelwas removed by centrifugation to separate the supernatant. Eachsupernatant was assayed for hemagglutination activity and endotoxincontent by the methods described below. The results are shown in Table3.

[0056] Determination of hemagglutination activity: After the sample wasserially diluted 2 folds with a 0.01 M phosphate buffered saline, 0.6v/v% chick immobilized red blood cells were added to causehemagglutination. The maximum dilution rate of each sample showinghemagglutination was taken as the hemagglutinin titer HA. Determinationof endotoxin (ET) content: Using Escherichia coli (Difico 055-B5) as areference strain, ET content was determined by the Limulus test (WakoPure Chemical kit). Results are expressed in ng/ml unit. It is evidentfrom Table 3 that endotoxin can be selectively removed, without activeingredient loss, by treating the sample with previously preparedaluminum hydroxide gel in the presence of ammonium sulfate. TABLE 3Amount of Ammonium Endotoxin Sulfate Added Content HA Value HA Recovery(w/v %) (ng/ml) (HAU/ml) Rate* (%) 0 15.8 16000 50.0 2 11.1 32000 100.04 <9.0 32000 100.0 8 <9.0 24000 75.0

Example 2

[0057] To each of pertussis toxin (PT), pertussis filamentoushemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussis fimbriae(FIM) as obtained in Example 1, a half amount of a saturated ammoniumsulfate solution was added, followed by sufficient stirring. After beingkept standing at 4° C. for 1 week, the mixture was again centrifuged;the resulting precipitate was collected.

[0058] This precipitate was dissolved in a 0.025 M phosphate buffer (pH7.0) supplemented with 0.25 M sodium chloride to yield a solution ofpertussis toxin (PT), pertussis filamentous hemagglutinin (FHA),pertactin (PRN, 69K-OMP) or pertussis fimbriae (FIM). To each solution,a saturated ammonium sulfate solution was added to a final concentrationof 4.0 v/v%. To this mixture, previously prepared, recovered aluminumhydroxide gel was added to a final concentration of 0.4 mg/ml, followedby gentle stirring for 30 minutes at room temperature. After completionof the reaction, the aluminum hydroxide gel was removed bycentrifugation to yield pertussis toxin (PT), pertussis filamentoushemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussis fimbriae(FIM).

[0059] Pertussis toxin (PT) content, pertussis filamentous hemagglutinin(FHA) content, pertactin (PRN, 69K-OMP) content and pertussis fimbriae(FIM) content were determined in the same manner as in Example 1; andendotoxin content, in the same manner as in Reference Example 1. Theresults are shown in Table 4. TABLE 4 Endotoxin Content ActiveIngredient (ng/100 μg Protein Content Recovery Sample protein) (μgprotein/ml) Rate* (%) PT 0.01 2999.0 82.5 FHA 0.11 9060.2 95.1 FIM 0.54478.6 70.9 69K-OMP 0.08 3505.8 80.9

[0060] It is evident from this table that endotoxin was selectivelyremoved, with substantially no loss of any protective component, theendotoxin content per 100 μg protein/ml being not more than 1 ng/ml forall components.

Example 3

[0061] To each of pertussis toxin (PT), pertussis filamentoushemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussis fimbriae(FIM) as obtained in Example 1, a half amount of a saturated ammoniumsulfate solution was added, followed by sufficient stirring. After beingkept standing at 4° C. for 1 week, the mixture was again centrifuged;the resulting precipitate was collected. This precipitate was dissolvedin a 0.05 M phosphate buffer (pH 8.0) supplemented with 1 M sodiumchloride, after which it was dialyzed by the tube method using a 0.05 Mphosphate buffer (pH 8.0) supplemented with 1 M sodium chloride as theexternal fluid, to yield a solution of pertussis toxin (PT), pertussisfilamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) or pertussisfimbriae (FIM). The concentrate dialyzate was subjected to sucrosegradient density centrifugation on a sucrose density gradient of 1-30w/w% and at an R_(max) of 64,900 G for about 18 hours. After completionof the centrifugation, 34 w/w% sucrose was fed into the rotor at a lowrate of rotation to collect fractions.

[0062] Pertussis toxin (PT) content, pertussis filamentous hemagglutinin(FHA) content, pertactin (PRN, 69K-OMP) content and pertussis fimbriae(FIM) content were determined in the same manner as in Example 1; andendotoxin content, in the same manner as in Reference Example 1. Theresults are shown in Table 5. TABLE 5 Endotoxin Content ActiveIngredient (ng/100 μg Protein Content Recovery Sample protein) (μgprotein/ml) Rate* (%) PT 0.04 231.2 82.5 FHA 0.01 849.4 79.3 FIM 0.2049.1 80.3 69K-OMP 0.03 319.8 92.0

[0063] It is evident from this table that endotoxin was selectivelyremoved, with substantially no loss of any protective component, theendotoxin content per 100 μg protein/ml being not more than 1 ng/ml forall components.

Example 4

[0064] To the PT as obtained in Example 3, with addition of amino acidsuch as Lysine, was added formalin to a final concentration of 0.4 v/v%, and after through mixing, was allowed to stand in an incubator at 39°C. for 21-35 days.

[0065] To each of FHA, 69K-OMP and FIM as obtained in Example 3, wasadded formaline to a final concentration of 0.4 v/v %, and after throughmixing, was allowed to stand in an incubator at 39° C. for 7 days.

[0066] Each of these components as treated above was dialyzed against 4mM phosphate buffer (pH 7.0) supplemented with 0.15M sodium chloride toyield detoxificated PT, inactivated FHA, inactivated 69K-OMP andinactivated FIM.

[0067] These detoxificated or inactivated components were blended inseveral ratios shown in Table 6 and 7, and followed by addition ofaluminum chloride to a final concentration of 0.2 mg/ml to give avaccine respectively.

[0068] The results for the experiments of mouse intracerebral potencywith these blended vaccines, are shown in Table 6 and 7. The experimentswere performed according to the method of Japanese Minimum Requirementsfor Biological Products (Association of Biologicals Manufactures ofJapan). TABLE 6 Mouse intracerebral Protein content of respectivepotency protective components 50% effective (μg protein/ml) dose PT FHAFIM 69K-OMP IU/ml (μg protein) 10 40 0 0 10.9 1.06 20 30 0 0 13.4 0.8920 40 0 0 11.6 1.18 20 50 0 0 13.6 1.18 20 80 0 0 12.6 1.81 30 40 0 019.3 0.85 40 40 0 0 18.7 1.04

[0069] TABLE 7 Mouse intravcerebral Potein content of respective potencyprotective components 50% effective (μg protein/ml) dose PT FHA FIM69K-OMP IU/ml (μg protein) 25 25 0  0 19.5 1.18 25 25 5  0 26.0 0.98 2525 0 10 24.1 1.18 25 25 5 10 19.3 1.60 25 50 0  0 24.1 1.47 25 50 5  022.2 1.70 25 50 0 10 23.7 1.68 25 50 5 10 24.8 1.71

[0070] It is evident from these figures that both inactivated 69K-OMPand inactivated FIM had small effects on the mouse intracerebralpotency, and no significant difference were observed among the blendedvaccines which contain more than 25 μg protein/ml of detoxificated PT.

Example 5

[0071] The experiment of mouse aerozol infection protecting potency wereperformed with the blended vaccines as obtained in Example 4. Eachvaccine diluted to one-third was subcutaneously administered to 4week-old mouse respectively with 0.2 ml of each diluted one. Four weekslater after the administration, each mouse was subjected to airwayinfection with 18-323 phase I strain of Bordetella pertussis by usingthe aerozol chamber, and 10 days later after the infection, the abdomenof each mouse was opened and the trachea and lung were picked out fromeach infected mice.

[0072] The specinen of each homoginized tissue applied to Bordet-Gengouagar. The agar was cultured at 35° C. for 5 days and the colonies ofBordetella pertussis were counted.

[0073] Based on the colony counts of the non-administered mice, theprotective dose was calculated.

[0074] The 75% protective dose was calculated in the case of trachea,and the 50% protective dose was calculated in the case of lung. Resultswere expressed in μg protein.

[0075] And growth inhibitory rate was calculated in the high doseadministered group. Equation of the growth inhibitory rate was asfollows.${{Growth}\quad {inhibitory}\quad {rate}\quad (\%)} = {\left( {1 - \frac{\begin{matrix}{{Colony}\quad {counts}{\quad \quad}{of}{\quad \quad}{High}\text{-}{dose}} \\{{administered}\quad {mice}}\end{matrix}}{\begin{matrix}{{Colony}\quad {counts}\quad {of}} \\{{non}\text{-}{administered}\quad {mice}}\end{matrix}}} \right) \times 100}$

[0076] Results are shown in Table 8. TABLE 8 Protein content ofrespective protective Trachea Lungs components 75% 50% (μg protein/ml)Protective Growth Protective Growth 69K- dose (μg inhibitory dose (μginhibitory PT FHA FIM OMP protein) rate (%) protein) rate (%) 40 40 0 03.75 97.5 1.00 89.9 20 80 0 0 4.76 87.7 1.10 92.6 25 50 0 0 4.14 89.50.96 89.7 25 50 5 0 1.07 100 0.49 100 25 50 0 10  1.74 100 0.49 100 2550 5 10  0.52 100 0.26 100

[0077] It is evident from this table that both inactivated 69K-OMP andinactivated FIM ahad small effects on the mouse intracerebral potency,but they showed protective effect on the aerozol infection potency.

Test Example 1

[0078] Differences of adsorption performance between the calciumphosphate gel (In-side Gel) forming and the hydroxyapatite gel (Out-sideGel) on FHA and FIM.

[0079] Roux bottle stationary culture prepared by method described inExample 1. The cell culture was concentrated to a one-tenth volume usingan ultrafiltration membrane, after which it was centrifuged to separatethe supernatant (Sample a) and cells. The cells were dispersed in aone-tenth volume of 0.05 M phosphate buffer (pH 8.0) supplemented with 1M sodium chloride and stirred well. It was kept standing at 4° C. for 4days, followed by centrifugation to yield the eluted supernatant (Sampleb) included PT, FHA, 69K-OMP and FIM.

[0080] The culture supernatant (Sample a) and eluted supernatant (Sampleb) described above were treated as following 1) or 2).

[0081] 1) Calcium Phosphate Gel (In-side Gel) Forming Treatment

[0082] To the samples, a 1 M phosphate buffer (pH 8) was added, followedby addition of a calcium acetate solution to a final concentration of0.5w/v%, 1.0w/v% or 2.0 w/v% and gently stirred at room temperature for1 hour, followed by centrifugation at 1000 rpm for 10 minutes tosupernatant and gel residue. The eluted solution was obtained by elutingthe gel residue with a 0.1 M phosphate buffer (pH 8.0) supplemented witha 1M sodium chloride.

[0083] 2) Hydroxyapatite Gel (Out-side Gel) Treatment

[0084] Hydroxyapatite gel (produced by BDH Chemicals Ltd) wasequilibrated with a 0.01M phosphate buffer (pH 8.0). The gel was addedto 20w/v%, 10w/v% or 50w/v% to the sample volume and gently stirred atroom temperature for 1 hour, followed by centrifugation at 1000 rpm for10 minutes to seperate supernatant from gel residue. The eluted solutionwas obtained by eluting the gel residue with a 0.1 M phosphate buffer(pH 8.0) supplemented with a 1M sodium chloride.

[0085] The content of FHA or FIM cotent in each sample was determined byELISA, with FHA or FIM as the house references. The assay results areexpressed in μg protein/ml unit. Protein content; Protein precipitatedwith heated trichloroacetic acid was quantitated by the Lowry method,with bovine serum albumin (Fraction V, produced by Wako Pure ChemicalIndustries) as a refference. Results are expressed in μg protein/mlunit.

[0086] Adsorption rate and recovery rate to the gel on FRA and FIM werecalculated by following equations respectively.${{Adsorption}\quad {rate}\quad (\%)} = {\left( {1 - \frac{\begin{matrix}{{Supernatant}\quad {of}} \\{{post}\text{-}{gel}\quad {treatment}}\end{matrix}}{\begin{matrix}{{Pre}\text{-}{gel}\quad {treatment}} \\{Sample}\end{matrix}}} \right) \times 100}$${{Recovery}\quad {rate}\quad (\%)} = {\frac{\begin{matrix}{{Eluted}\quad {solution}\quad {of}} \\{{post}\text{-}{gel}\quad {treatment}}\end{matrix}}{\begin{matrix}{{Pre}\text{-}{gel}\quad {treatment}} \\{Sample}\end{matrix}} \times 100}$

[0087] Results are shown in Table 9 and Table 10 TABLE 9 a) Culturesupernatant FHA Absorp- FIN tion Recovery Absorption Recovery rate (%)rate (%) rate (%) rate (%) Concentration 0.5 89.7 69.6 0.0 0.0 ofcalcium 1.0 90.4 63.0 95.0 77.9 acetate added 2.0 89.5 44.4 94.3 98.4(w/v %) Concentration 2.0 25.4 27.5 0.0 0.0 of 10.0 49.5 26.4 7.9 4.1hydroxyapatite 50.0 90.8 48.9 22.1 5.3 added (w/v %)

[0088] TABLE 10 b) The eluted solution from the cell with 0.05Mphosphate buffer (pH 8.0) supplemented with 1M-NaCl FHA Absorp- FIN tionRecovery Absorption Recovery rate (%) rate (%) rate (%) rate (%)Concentration 0.5 96.3 87.2 18.7 12.5 of calcium 1.0 98.7 61.0 99.9 74.3acetate added 2.0 98.7 55.1 99.8 94.3 (w/v %) Concentration 2.0 10.8 1.04.6 1.1 of 10.0 4.7 3.8 9.7 3.9 hydroxyapatite 50.0 15.6 16.7 13.5 8.2added (w/v %)

[0089] The calcium phosphate gel (In-side gel) strongly adsorbs both FHAand FIM, but the hydroxyapatite gel has small adsorption effect on theFIM. Also the Hydroxyapatite gel compared with the calcium phosphategel, has less adsorption effect on the FHA and depend on the volumeadded.

[0090] Industrial Applicability

[0091] The method of the present invention is characterized by the useof the same means of purification for all subject protective componentsof Bordetella pertussis. Each component can therefore be purified withhigh efficiency and high recovery rate, an aspect very advantageous forindustrial production. It is also possible to efficiently produce animproved purified pertussis component vaccine comprising an effectivecombination of pertussis filamentous hemagglutinin (FHA), pertactin(PRN, 69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT).

1. A method of separating at least one member of the group consisting ofpertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP),pertussis fimbriae (FIM), and pertussis toxin (PT) by bringing aBordetella pertussis culture into contact with calcium phosphate gelwhich is formed by adding calcium ions to the culture in the presence ofphosphate ions.
 2. A method of separating at least one member of thegroup consisting of pertussis filamentous hemagglutinin (FHA), pertactin(PRN, 69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT) byseparating a Bordetella pertussis culture into cells and culture liquid,and carrying out at least one of processes (A), (B), (C) and (D): (A) aprocess in which the separated cells are eluted with a salt solution,and pertussis filamentous hemagglutinin (FHA) is separated by bringingthe eluted solution into contact with calcium phosphate gel of claim 1,(B) a process in which the cell residue resulting from the elutiontreatment of the above process (A) is heated in the presence of a saltsolution and brought into contact with calcium phosphate gel, andpertactin (PRN, 69K-OMP) is separated by bringing the eluted solutioninto contact with calcium phosphate gel of claim 1, (C) a process inwhich the cell residue resulting from the elution treatment of the aboveprocess (A) is heated in the presence of a salt solution, thesupernatant is brought into contact with calcium phosphate gel andeluted with a salt solution, and pertussis fimbriae (FIM) is separatedby bringing the eluted solution into contact with calcium phosphate gelof claim 1, (D) a process in which the culture or the separated cultureliquid is brought into contact with calcium phosphate gel of claim 1,and pertussis toxin (PT) is separated from the supernatant.
 3. Theseparation method of claim 2, wherein the supernatant is brought intocontact with calcium phosphate gel and eluted with a salt solution toseparate pertussis filamentous hemagglutinin (FHA) in process (A). 4.The separation method of claim 2, wherein the supernatant after beingbrought into contact with calcium phosphate gel is brought into contactwith ion exchange gel to separate pertactin (PRN, 69K-OMP) in process(B).
 5. The separation method of claim 2, wherein the supernatant isbrought into contact with calcium phosphate gel and removed, and theresulting residue is eluted with a salt solution to separate pertussisfimbriae (FIM) in process (C).
 6. The separation method of claim 2,wherein the supernatant is brought into contact with ion exchange gel toseparate pertussis toxin (PT) in process (D).
 7. The separation methodof claim 2, wherein the salt solution used in processes (A) and (C) is abuffer containing an alkali metal salt.
 8. The separation method ofclaim 7, wherein the salt solution is a buffer containing 0.01-1.0 Msodium chloride.
 9. The separation method of claim 1 or 2, wherein thecalcium phosphate gel is formed by adding calcium ions to the culture orthe supernatant of pH 7-9 in the presence of phosphate ions.
 10. Theseparation method of claim 9, wherein the equivalent ratio of phosphateions and calcium ions is 1.25-30 equivalents of phosphate ions perequivalent of calcium ions.
 11. The separation method of claim 9,wherein the calcium phosphate gel is formed by adding calcium acetate,as a calcium ion source, at 0.1-2 w/v% in the presence of a 0.05-0.1 Mphosphate buffer.
 12. The separation method of claim 1 or 2, wherein atleast one member of the group consisting of pertussis toxin (PT),pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) andpertussis fimbriae (FIM) is separated, after which endotoxin is removedby adsorption to aluminum hydroxide gel in the presence of ammoniumsulfate.
 13. The separation method of claim 1 or 2, wherein at least onemember of the group consisting of pertussis toxin (PT), pertussisfilamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussisfimbriae (FIM) is separated, after which endotoxin is removed by zonalcentrifugation.
 14. A pertussis vaccine wherein the componentsPT:FHA:FIM are admixed in a ratio of 4-6:8-10:1.
 15. A pertussis vaccinewherein the components PT:FHA:PRN:FIM are admixed in a ratio of2-6:4-10:1-2:1.